| Objective: Construct the stable within macrophages(MΦ)expressing GFP integrated Brucella GFP-M5 and RFP integrated Brucella 16 M,respectively(Hereinafter referred to as GFP-M5 and RFP-16M);the levels of TNF-α,IFN-γ and NO released by MΦ infected with GFP-M5 and RFP-16 M at different concentrations of IL-2 and at different time points were detected;CFU was used to determine the viable count of GFP-M5 and RFP-16 M in MΦ,and the phagocytic index of MΦ was calculated to determine the phagocytosis of MΦ and its bactericidal ability;the phagocytosis rate of MΦ was calculated by counting the number of fluorescent cells by using a hemacytometer and inverted fluorescence technique;trypan blue staining to detect the survival rate of MΦ after being infected with different concentrations and time points;under the action of IL-2 at different time points,the proliferation of MΦ was observed under a light microscope using a hemocytometer;to detect the contents of secretion of IL-2 MΦ itself by using RT-PCR kit,judgment of exogenous IL-2 the adjustment role of experiment;to investigate the effect of IL-2 on the immunomodulation of MΦ after being infected with GFP-M5 and RFP-16 M,and to provide a new idea and theoretical basis for the clinical use of IL-2 and the treatment of brucellosis.Methods: 1、 The normal RFP-16 M and RFP-16 M culture to the logarithmic phase after the collection of bacteria,with Microblogging turbid tube configuration of the desired concentration of bacteria,bacteria and cells in accordance with the ratio of 100: 1 infection,the use of plate count France to compare the normal bacterial RFP-16 M and RFP-16 M in the intracellular survival ability;2、 The levels of TNF-α,IFN-γ and NO released by infected macrophages were detected by ELISA kit and microplate reader under different concentrations of IL-2 and different time points,respectively;3、 According to the method of colony forming unit(CFU),infected cells with different concentrations of IL-2 and different time points were lysed with cell lysate,diluted in PBS and then plated on Brucella solid medium for 72 h After colony counting,calculate the concentration,the number of colonies at each time point and the proportion of cells,get MΦ phagocytic index;4、 Under the plate of hemocytometer and light microscope,the proliferation of infected MΦ was observed under different concentrations of IL-2 and different time points;the cell death rate was calculated by trypan blue staining,and the survival rate of M was calculated;5、 The phagocytosis rate of MΦ was calculated by counting the number of luminescent cells and the total number of cells according to the characteristics of GFP-M5 and RFP-16 M expressing fluorescent protein only in MΦ cells using hemacytometer and laser scanning confocal microscope;6、 The MΦ RNA was extracted and the template c DNA was reverse transcribed by RT-PCR kit.The c DNA was amplified by PCR,and observe whether the gene sequence of M IL-2 is transcribed to express m RNA.Results: 1、 The results of CFU showed that there was no significant difference in the counts of bacteria in different stages of RFP-16 M Brucella isolates of RFP-16 M compared with that of the normal strains.The two strains were transformed with p MC-221 vector and infected with mouse macrophages the infectivity did not affect;IL-2 promoted the ability of MΦ to phagocytose RFP-16 M.With the increase of infection time and the increase of IL-2 concentration,the number of intracellular viable cells decreased in a time-and concentration-dependent manner,indicating that MΦ(IL-2 was 0 × 106 IU,the number of colonies was 858.667 ± 48.222 at 0h;the concentration of IL-2 was 5 × 106 IU at 24 h,the number of colonies was 285.667 ± 7.572)Under the regulation of IL-2,phagocytic index and phagocytic rate of phagocytosis of RFP-16 M by MΦ were significantly different from that of blank control group(P <0.05),and decreased as a whole The objective is to prove that IL-2 promotes the activation of macrophages and enhances its bactericidal and immunomodulatory capacity;2、 The levels of cytokines(TNF-α,IFN-γ and NO)released by MΦ infected with GFP-M5 by IL-2 were detected by ELISA kit using ELISA kit according to the instructions of microplate reader(GFP-M5 invasion Staining,IL-2 at 5 × 106 IU,12 h vs.0 h: 123.902 ± 13.249 vs 11.092 ± 3.152;67.083 ± 26.675 vs 5.346 ± 0.835;14.779 ± 1.899 vs.3.256 ± 0.269)were significantly increased compared with the control group(P <0.05).The concentration of IFN-γ and NO released by RFP-16 M cells were significantly lower than those of blank control group(96.667 ± 10.786 and 22.333 ± 3.055;3.140 ± 0.403 and 1.511 ± 0.344)(P <0.05).Compared with the blank control group,the level of TNF-α in serum was significantly increased(103.704 ± 7.407 and 370.370 ± 147.347)(P <0.05).Through CFU counting,it was found that IL-2 promoted the phagocytosis of RFP-16 M by macrophages.With the increase of infection time and IL-2 concentration,the intracellular viable cells decreased in a time-and concentration-dependent manner the ability of phagocytosis of GFP-M5 by macrophages was enhanced to a certain extent.With the increase of IL-2 concentration,the number of intracellular viable cells also increased.The concentration of IL-2 Dependency;3、 Under normal conditions,the number of MΦ gradually increased with the increase of culture time(T≤24h).After the infection with RFP-16 M,MΦ increased at 2h with the increasing of IL-2 concentration,(P <0.05),while the rest of the time points showed a downward trend,which was significantly different from the control group(P <0.01).Under the same concentration regulation,The increase of infection time showed a trend of decreasing and then increasing(IL-2 was 3 × 106 IU,0h,2h,398.333 ± 10.408 at 12 h,518.333 ± 17.559;476.667 ± 7.638)GFP-M5 infected MΦ cultured 2h,6h,the concentration of IL-2 regulation,phagocytosis of MΦ phagocytosis GFP-M5 were significantly different from the blank group(P <0.01),indicating that under these conditions,MΦ The ability to phagocytose GFP-M5 is optimal;4、 Under phagocytosis of IL-2,phagocytic index and phagocytosis rate of phagocytosis of RFP-16 M by MΦ increased with infection time(IL-2 was 3 × 106 IU,12h,0.934 ± 0.081)and blank group(2.192 ± 0.123)(P <0.05),and showed an overall downward trend.It was proved objectively that IL-2 promoted the activation of MΦ and enhanced its bactericidal and immunomodulatory capacity.The m RNA of MΦ in normal growth condition was extracted and RT-PCR kit was used to reverse transcribe the template c DNA.MΦ itself did not secrete IL-2 by PCR and gel electrophoresis,indicating that the difference of the experimental results,mainly from the regulation of exogenous IL-2;5、 At the same time point of infection,the phagocytic index of phagocytic index of MΦ phagocytosis GFP-M5 was significantly different(P <0.05)at different concentrations of IL-2 compared with the blank group,and at 2h,IL-2 concentration At 5 × 106 IU,the phagocytic index reached the maximum [12.486 ± 0.339],higher than that of the adjacent two groups [4.458 ± 0.157 and 1.558 ± 0.076],which was significantly higher than that of the control group.Indicating that IL-2 immunomodulatory function with a two-way;MΦ phagocytosis had a significant role in promoting,and there concentration and time-dependent;6、 Trypan blue staining showed that the number of MΦ infected by RFP-16 M was significantly decreased at different concentrations(P <0.01)as the infection time increased;7、 The m RNA of MΦ in normal growth condition was extracted and RT-PCR kit was used to reverse transcribe the template c DNA.MΦ itself did not secrete IL-2 by PCR and gel electrophoresis,indicating that the difference of the experimental results,mainly from the regulation of exogenous IL-2.Conclusion: The results of this study show that macrophages do not secrete IL-2 itself.The differences in the results of this experiment are mainly due to the regulatory effect of exogenous IL-2.IL-2 can promote the activation of MΦ and stimulate MΦ to enhance its ability to secrete TNF-α,NO and IFN-γ,so as to participate in the anti-Brucella immune response and thus provide IL-2 research and treatment for brucellosis New ideas and theoretical basis;IL-2 can enhance phagocytosis and bactericidal ability of macrophages to a certain extent,and there is a concentration and time-dependent manner.IL-2 also has bi-directional regulation at both concentration and time points,Due to the phagocytosis and bactericidal ability of the cells,the role of lysosomes in the immune response is also enhanced,indirectly resulting in cell proliferation and survival also affected by IL-2 concentration and Brucella infection. |
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